The present invention relates to novel immunological reagents and their use in assays for the detection of kidney disease or damage (hereinafter also sometimes called "nephropathy"), and, in particular, assays capable of detecting nephropathy at developmental stages not possible to detect with current methodologies. Fundamental to the present invention is the discovery of a human nephropathy-related protein which is a glycoprotein in the immunoglobulin class of globular proteins and which has been demonstrated to be associated with development of overt kidney disease. This nephropathy-related immunoglobulin-like protein (hereinafter referred to as "NRIg") is believed to be capable of serving as a very early and specific marker for kidney-related diseases.
Thus, in one embodiment, the present invention relates to the isolation and purification of NRIg from human body fluids using the immune complex-capturing protein reagent described as "RhC" in U.S. Pat. No. 4,783,528.
In another embodiment, the invention relates to the use of NRIg in its purified form for the preparation of highly specific antibodies for use in the assays of the present invention. An especially preferred embodiment of the present invention relates to the use of such antibodies, and immobilized RhC, in a unique and highly sensitive immunologic assay for detecting and/or determining the concentration of NRIg in urine as a predictor of the development of overt nephropathy.
In yet another embodiment, the present invention provides simple, economical, and reliable assays for the detection of NRIg in biological fluids. These assays utilize the monoclonal antibodies of this invention. Because of their specificity for NRIg, these antibodies are particularly useful for the detection of kidney disease in a wide variety of clinical settings by a variety of methods ranging, for example, from sophisticated enzyme-linked immunosorbant assays (EIA) to elegant and simple radial immunodiffusion (RID) assays.
The immunologic terms employed herein are believed to be in accord with conventional usage and definition. Should any presently unforeseen confusion arise, unless otherwise indicated, the construction of a term shall be in accordance with its definition and usage in the well known textbook by William E. Paul, Fundamental Immunology, second edition (1989), Raven Press Ltd, New York.
For the convenience of the reader, several publications are referenced in the present discussion. While these references will more fully describe the state of the art to which the present invention pertains, the inclusion of these references is not an admission that any represent prior art with respect to the present invention.
The clinical diagnosis of kidney disease as currently practiced relies almost exclusively on observed changes in levels of blood urea nitrogen (BUN) and of creatinine in plasma, as well as on abnormalities detected in urinalysis, and on measured changes in urine output. While these tests are useful in revealing existing renal damage, they are not useful in predicting future kidney damage.
The present discussion will serve to illustrate the difficulties which clinicians and research investigators alike face in accurately detecting kidney disease very early in its development, i.e., in its incipient form, not only in individual patients without a history of intrinsic renal disease, but especially in those patients whose systemic illness will likely, at some point during the course of their disease, affect the kidneys.
The underlying pathogenic mechanism(s) of most kidney disease is not known, in spite of the wide occurrence of this clinical phenomenon, and despite extensive clinical and laboratory investigation into the problem. Given the wide occurrence of pathological conditions which may affect the kidneys, it is surprising that there still is no single noninvasive diagnostic test of a measureable substance which can be used to predict with confidence which individuals will develop nephropathy as a complication of their primary disease. Such a diagnostic test for incipient nephropathy should constitute an "early warning signal," when medical intervention could still limit the extent of subsequent kidney damage.
Toward this end, much effort has been devoted to the measurement of low-molecular weight plasma proteins, such as beta-2-microglobulin in the urine or serum, or to the determination of a variety of renal enzymes, such as L-alanine aminopeptidase (AAP) and N-acetyl-beta-D-glucosaminidase (NAG) which are released into the urine when renal injury occurs. Although these tests have provided much useful information, they have had limited application, not only because they are nonspecific with regard to the cause and site of renal injury, but also because the test proteins are not very stable in urine. Furthermore, a variety of enzyme inhibitors and other interfering materials are usually present in urine, and this significantly reduces the specificity and usefulness of these tests.
To improve the usefulness of noninvasive tests, several investigators have developed immunologic measurements of specific renal antigens present in the urine in order to determine not only the occurrence of nephropathy, but also the site and severity of the renal injury. For example, Schoenfeld and Glassock (Kidney Intl 3: 309-314, 1973) developed an immunodiffusion technique to identify a proximal tubule brush-border epithelial antigen in concentrated human urine. Zager and colleagues extended this work by developing a radioimmunoassay for a proximal renal tubular epithelial antigen (termed HRTE-1) in urine. The assay (Nephron 26: 7-12, 1980) lacked specificity, however, because this antigen could be detected in organs other than kidney.
A number of investigators have reported on the use of monoclonal antibodies as probes for detecting the presence of specific kidney-related antigens in serum and urine (Sachse et al., Clin Chim Acta 110: 911104, 1981; Michael et al., Kidney Intl 24: 74-86, 1983; Tolkoff-Rubin, Kidney Intl.29: 142-152, 1986). The use of these antibodies provided a quantitative means of determining both the site and degree of nephropathy. Because serial samples of urine are readily obtained, this approach permitted monitoring the stage and activity of kidney diseases, as well as its responsiveness to therapy. For example, the use of monoclonal antibodies to detect the 120 kDa proximal tubule antigen, adenosine deaminase binding protein (ABP), which is released from renal brush border epithelial cells into the urine in renal disease, has been described in U.S. Pat. No. 4,731,326. The shortcoming of a number of these assays for detecting the presence of renal proteins in urine, however, is that the antigens being detected are not useful as very early predictors of incipient kidney disease; rather, the assays are useful primarily in determining the type and location of clinically overt nephropathy.
In the group of patients with diabetes mellitus, approximately 35 percent of patients will develop kidney disease and progress to end-stage renal disease approximately 15 years after the onset of diabetes. Unfortunately, the kidney disease in these individuals is usually very advanced and irreversible by the time it is diagnosed by the markers currently being used, which are the persistent presence of an excess of serum proteins (such as albumin) in the urine, and an elevated level of creatinine in the blood.
"Albustix"-positive proteinuria in the urine of diabetic patients, a condition often referred to as macroproteinuria or macroalbuminuria, normally signals the presence of overt clinical nephropathy. Many of the patients who exhibit this advanced-stage of kidney dysfunction also have elevated blood pressure. Kidney biopsies taken at this stage in the disease process usually show advanced and irreversible structural damage to the kidney, and it is very likely that this damage was present in the kidney long before it was detected clinically by the occurrence of proteinuria.
In contrast to this late detection of macroproteinuria, some investigators are advancing the notion that the detection in urine of very small amounts of albumin (microalbuminuria) is a more sensitive marker for kidney disease (e.g., see Mogensen C E. Diabetes 39: 761-767, 1990). Unfortunately, exercise, poor control of blood sugar levels, and other metabolic imbalances can nonspecifically increase the albumin excretion in diabetic subjects, thereby reducing the clinical value of microalbuminuria as a reliable single marker of early kidney disease in these individuals. Furthermore, strenuous exercise in itself is sufficient to induce microalbuminuria in the urine of some healthy individuals.
It would be of great clinical benefit to have a test for detecting a substance that is associated specifically with the beginning stages in the development of nephropathy, even before the renal damage becomes a clinical problem. Such early detection of nephropathy is critically important so that appropriate therapy can be initiated in these patients before significant kidney damage has occurred. When kidney disease has been detected early in experimental groups, successful therapy has resulted in the stabilization of progressive kidney disease. Clearly, the very great benefits to be derived from early detection of nephropathy within defined clinical populations mandates the development of detection systems more specific than microalbuminuria.
The potential for the use of the NRIg as an early marker for kidney disease is highly significant. The test for a protein marker other than albumin in the urine of patients as an indicator of the early development of kidney disease would enable the physician to differentiate between the initiation of kidney disease and exercise-induced or incidental albuminuria that can occur sporatically in patients with any of a variety of diseases. Also, a specific marker for end-stage renal disease would be highly beneficial for monitoring and evaluating treatment modalities in clinical trials, and would also identify high-risk patients who would benefit from such therapies.